Muscle fatigue, defined as an inability to maintain a constant work intensity, has not been adequately explained on the cellular level. No matter how fatigue is produced (by extended repetition, ischemia, or hypoxia), it is accompanied by a qualitatively similar (though not identical) set of changes in the intracellular milieu. Our working hypothesis is that these changes in milieu are responsible for fatigue by causing major depressant effects on both the excitation-contraction coupling (ECC) process and the contractile apparatus (CA) of skeletal and cardiac muscle. Our work to date demonstrates that the net effect of milieu changes is to decrease the maximum force and calcium sensitivity of the CA. This net effect, the non-linear summation of the effects of changes in a number of intracellular constituents, does not totally explain the severe loss of force observed in intact, isolated muscles. The purpose of this proposal is to test the hypothesis in a two-fold manner: A) To complete the present series of experiments on skeletal and cardiac muscle: 1) by determining the dependence of the properties of the CA on free energy from ATP. 2) by determining whether the CA is sensitive to ionic strength or ionic equivalence as a measure of the intracellular ionic environment. B) To extend the scope of our studies to determine how the changes in milieu associated with fatigue affect. 1) the maximum velocity of shortening (Vmax - measured by the slack test). 2) the response to myosin light chain phosphorylation. 3) the ability of the sarcoplasmic reticulum (SR) to sequester and release calcium. 4) the ability of inositol trisphosphate (IP3), a possible second messenger, to modulate calcium-induced release of calcium from the SR. This project is unique in that it offers a unified and systematic approach to the problem of fatigue on the cellular level. Only such a study can: 1) determine the interactive effects of changing intracellular constituents (e.g. pH and Pi) to produce the net effect; 2) accurately compare properties of skeletal and cardiac fibers (e.g. the depressant effect of changes in phosphocreatine is greater in heart than in skeletal muscle). The proposed experiments will address both basic and clinical questions, the intrinsic mechanism of muscle contraction and the causes of its failure with fatigue.